Nuclear protein import is a fundamental, but poorly understood cellular process. All intranuclear processes require imported proteins and a number of these processes (DNA replication, transcription of specific genes, cell cycle events etc.) are regulated by the timing of import of specific proteins. Nuclear protein import is an energy-dependent, signal- mediated process. While the protein nuclear localization sequences (NLSs) required for import are relatively well-defined, the molecular mechanisms by which these signals trigger translocation of the import substrate through the nuclear pore complex (NPC) into the nuclear interior remain unclear. Four soluble proteins have been identified that are required to support nuclear protein import in digitonin permeabilized cells: Ran, p10, karyopherin beta, and importin (which is probably equivalent to karyopherin alpha). Ran is a member of the Ras superfamily of small GTP- binding proteins and is the only member of this family to be localized primarily inside the nucleus. In addition to nuclear protein import, defects in Ran and its guanine nucleotide exchange factor RCC1 have also been shown to affect cell cycle progression, DNA replication, nuclear structure, and RNA transcription, processing, and export. Whether these pleiotrophic effects are all the result of import defects or whether Ran or RCC1 may have functions in addition to nuclear import is at present unclear. The observation that mutant forms of these proteins also affect mRNA export from the nucleus may be significant as it could mean that nuclear import and export are mechanistically similar. With the eventual goal of understanding the molecular mechanisms of nuclear protein import, three specific aims are proposed. The first is to determine whether a mixture of the four known import factors (in the form of purified recombinant proteins) is sufficient to support nuclear protein import in digitonin-permeabilized cells or whether there are additional cytosolic factors. If there are additional factors, they will be purified. The second specific aim is to determine how the GTPase cycle of Ran is related to the process of nuclear import and how it is modulated by other components of the transport machinery. The effects of the other soluble import factors and NPC components on guanine nucleotide binding and exchange and GTP hydrolysis by Ran will be measured. A GTPase defective mutant form of Ran will be tested for its effects on transport. The third specific aim is to determine whether the soluble import factors enter the nucleus in association with the substrate. A variety of techniques will be used to address this question including immunolocalization and immunoprecipitations and the tracking of labelled import factors during import.